TransDecoder (Find Coding Regions Within Transcripts)

TransDecoder identifies candidate coding regions within transcript sequences, such as those generated by de novo RNA-Seq transcript assembly using Trinity, or constructed based on RNA-Seq alignments to the genome using Tophat and Cufflinks.

TransDecoder identifies likely coding sequences based on the following criteria:

a minimum length open reading frame (ORF) is found in a transcript sequence

a log-likelihood score similar to what is computed by the GeneID software is > 0.

the above coding score is greatest when the ORF is scored in the 1st reading frame as compared to scores in the other 5 reading frames.

if a candidate ORF is found fully encapsulated by the coordinates of another candidate ORF, the longer one is reported. However, a single transcript can report multiple ORFs (allowing for operons, chimeras, etc).

optional the putative peptide has a match to a Pfam domain above the noise cutoff score.

By default, TransDecoder.LongOrfs will identify ORFs that are at least 100 amino acids long. You can lower this via the -m parameter, but know that the rate of false positive ORF predictions increases drastically with shorter minimum length criteria.

The process here is identical to the above with the exception that we must first generate a fasta file corresponding to the transcript sequences, and in the end, we recompute a genome annotation file in GFF3 format that describes the predicted coding regions in the context of the genome.

Construct the transcript fasta file using the genome and the transcripts.gtf file like so:

Next, convert the transcript structure GTF file to an alignment-GFF3 formatted file (this is done only because our processes operate on gff3 rather than the starting gtf file - nothing of great consequence). Convert gtf to alignment-gff3 like so, using cufflinks GTF output as an example:

Sample data and execution

The sample_data/ directory includes a runMe.sh script that you can execute to demonstrate the entire process, starting from a cufflinks GTF file. Note, the typical use-case for TransDecoder is starting from a fasta file containing target Transcripts, however, in the case of genome analysis, transcripts are often inferred from annotation coordinates, such as in this Cufflinks GTF formatted file. In this example, transcript sequences are reconstructed based on the GTF annotation coordinates, and then TransDecoder is executed on that fasta file. We include an additional utility for converting the transcript ORF coordinates into genome-coordinates so these regions can be examined in the genomic context.

Output files explained

A working directory (ex. transcripts.transdecoder_dir/) is created to run and store intermediate parts of the pipeline, and contains:

longest_orfs.pep : all ORFs meeting the minimum length criteria, regardless of coding potential.
longest_orfs.gff3 : positions of all ORFs as found in the target transcripts
longest_orfs.cds : the nucleotide coding sequence for all detected ORFs

longest_orfs.cds.top_500_longest : the top 500 longest ORFs, used for training a Markov model for coding sequences.

hexamer.scores : log likelihood score for each k-mer (coding/random)

longest_orfs.cds.scores : the log likelihood sum scores for each ORF across each of the 6 reading frames
longest_orfs.cds.scores.selected : the accessions of the ORFs that were selected based on the scoring criteria (described at top)
longest_orfs.cds.best_candidates.gff3 : the positions of the selected ORFs in transcripts

Then, the final outputs are reported in your current working directory:

transcripts.fasta.transdecoder.pep : peptide sequences for the final candidate ORFs; all shorter candidates within longer ORFs were removed.
transcripts.fasta.transdecoder.cds : nucleotide sequences for coding regions of the final candidate ORFs
transcripts.fasta.transdecoder.gff3 : positions within the target transcripts of the final selected ORFs
transcripts.fasta.transdecoder.bed : bed-formatted file describing ORF positions, best for viewing using GenomeView or IGV.

Including homology searches as ORF retention criteria

To further maximize sensitivity for capturing ORFs that may have functional significance, regardless of coding likelihood score as mentioned above, you can scan all ORFs for homology to known proteins and retain all such ORFs. This can be done in two popular ways: a BLAST search against a database of known proteins, and searching PFAM to identify common protein domains. In the context of TransDecoder, this is done as follows:

The outputs generated above can be leveraged by TransDecoder to ensure that those peptides with blast hits or domain hits are retained in the set of reported likely coding regions. Run TransDecoder.Predict like so:

The final coding region predictions will now include both those regions that have sequence characteristics consistent with coding regions in addition to those that have demonstrated blast homology or pfam domain content.

Viewing the ORF predictions in a genome browser

GenomeView or IGV are recommended for viewing the candidate ORFs in the context of the genome or the transcriptome. Examples below show GenomeView in this context.